1. Purified Nop1p and Rnt1p are able to process pre‐U18 molecules.
Purified Nop1p and Rnt1p are able to process pre‐U18 molecules. (A) Schematic representation of the int/U18wt construct utilized: the U18 coding region plus the flanking intronic regions containing the external stem were cloned under the T7 promoter. The transcribed RNA is 204 nucleotides long. (B) In vitro cleavage of the int/U18wt RNA (lanes U18wt) and of its mutant derivative in the box C (lanes U18bC) (see construct bC in Figure 3). 32P‐labeled RNAs were incubated in a 10 μl reaction with increasing concentrations of GST–Rnt1p protein (0.05 ng, lanes 2 and 5; 0.5 ng, lanes 3 and 6; 1 ng, lanes 4 and 7). In lanes 5–7, 10 ng of GST–Nop1p protein were added. Lanes 1: input RNA; lanes 8: RNAs were incubated only with GST–Nop1p (10 ng in 10 μl reaction). Incubations were allowed to proceed for 60 min. The RNA was extracted and run on a 6% polyacrylamide–urea gel and visualized by autoradiography. The products of the reaction are represented schematically at the side of the panel. (C) In vitro cleavage of a model snR38 precursor RNA. The 32P‐labeled int/38 RNA containing the snR38 snoRNA coding sequences plus the external stem and flanking intron sequences was treated as the RNA in (B). The int/snR38 RNA is 190 nucleotides long and the pre‐snR38 is ∼120 nucleotides long. (D) In vitro cleavage of 32P‐labeled snR190/U14dicistronic precursor RNA. The RNA transcript is the same as that used in Figure 4C. The reactions were performed as in (B). Reactions and lanes in (C) and (D) are numbered as in (B).
Corinna Giorgi et al. EMBO J. 2001;20:
© as stated in the article, figure or figure legend